Method for protecting the skin from aging

ABSTRACT

The present invention relates to a cosmetic treatment method for improving and/or protecting the human skin, scalp and/or mucous membrane, characterized in that a preparation containing at least one substance which modulates proteoglycans, more particularly lumican and/or syndecan and/or versican and/or decorin and/or glypican and/or biglycan, is topically applied. Such substances include plant extracts of  Pisum sativum  and  Vigna aconitifolia,  mannitol, cyclodextrin, yeast extract, disodium succinate, phytosterols, triterpenes, triterpene saponins, TGFβ, IL4, flavonoids, and flavonoid derivatives. The method can be applied for protecting the skin from aging and other detrimental influences of environmental pollutants and UV radiation, for boosting the functions of the dermal/epidermal junctions, and for improving wound healing.

FIELD OF THE INVENTION

This invention relates generally to cosmetic preparations and, moreparticularly, to a cosmetic process for protecting the human skin, scalpand/or mucous membrane against ageing, oxidative stress and the harmfuleffects of environmental toxins and UV radiation. The present inventionalso relates to the use of a substance which modulates lumican and/orsyndecan and/or versican and/or decorin and/or glypican and/or biglycanfor the production of cosmetic preparations and preparations fortreating particular skin diseases.

PRIOR ART

The extracellular matrix of the connective tissue consists of aplurality of macromolecules which form complex three-dimensionalnetworks, interact with one another and with the cells of the connectivetissue and thus contribute significantly to the structural integrity ofthe tissue. However, not only do they form a link between individualcells and tissues, they also provide for the regulation of cell growth,cell supply and cell differentiation through a filter and transportfunction (of growth factors, inhibitors or hormones) and through specialreceptor bonds. Key constituents of the extracellular matrix includecollagen, elastin, glycoproteins, hyaluronic acid, glycosaminoglycans,proteoglycans and glycoproteins which belong to the non-fibrous basicstructure.

Proteoglycans are macromolecules with a central protein to which one ormore glucosaminoglycan side chains are covalently bonded. They representthe main component in the extracellular matrix and comprise a number ofdifferent molecules which may be roughly divided into large and smallproteoglycans. Proteoglycans can be found at all levels of the basalmembrane which forms a largely homogeneous layer immediately below thebasal epithelial cells and which can be divided into three layers. Theuppermost part, which directly adjoins the cells, is the Lamina lucidawhich is followed by the Lamina densa as the middle layer of the basalmembrane and then by the outer Lamina fibroreticularis.

In the basal membrane, proteoglycans contribute towards tissue strengthvia the bonds to fibrous tissue constituents and, by binding water,influence the elasticity of the skin. Studies have shown that theincreased wrinkling of the skin and the reduction in elasticity isassociated with collagen fibers.

During ageing, the skin undergoes major changes in its mechanicalproperties, in its ability to retain water and in its firmness andelasticity. Proteoglycans have a major influence on these changes. Inaddition, in normal ageing processes, an opposite change is observed,i.e. a reduction in the large chondroitin sulfate proteoglycans(versican) and a parallel increase in the numbers of small dermatansulfate proteoglycans (decorin) with increasing age.

It is known that disease-induced changes in the skin can be influencedby changes in the proteoglycans. Thus, International patent applicationWO 01/17560 describes the treatment and prevention of bacterialinfections using substances which inhibit the release of syndecan-1.International patent application WO 94/12162 discloses the reduction intumour growth and the stimulation of hair growth by the stimulation ofsyndecan. In addition, it has been shown that scar formation is reducedor even prevented by administration of decorin, biglycan andfibromodulin (cf. International patent application WO 93/09800).

In view of the demand for preventing ageing-induced changes in the skinand for effective protection of the skin against environment-inducedageing effects, the problem addressed by the present invention was toprovide new mechanisms for improving the skin, scalp and/or mucousmembrane which would contribute to a delay in skin ageing and toprotection of the skin, scalp and/or mucous membrane againstenvironmental influences, oxidative stress, toxic substances or UVradiation and which could therefore be effectively used in cosmetic anddermopharmaceutical preparations for topical application.

DESCRIPTION OF THE INVENTION

The present invention relates to a cosmetic treatment process forimproving and/or protecting the human skin, scalp and/or mucousmembrane, characterized in that a preparation containing at least onesubstance which modulates proteoglycans, more particularly lumicanand/or syndecan and/or versican and/or decorin and/or glypican and/orbiglycan, is topically applied.

The present invention also relates to the use of a substance whichmodulates lumican and/or syndecan and/or versican and/or decorin and/orglypican and/or biglycan for the production of cosmetic preparations forprotecting the human skin, scalp and/or mucous membrane against ageing,for the production of cosmetic preparations for protection againstoxidative stress, for protection against toxic environmental influences,for protection against damage by UV light and for improving thefunctions of the dermal/epidermal junctions and to their use for theproduction of dermopharmaceutical preparations for improving the healingof wounds and preparations for treating alopecia, cellulitis orroseacea.

It has surprisingly been found that the modulation of proteoglycans,more particularly lumican, syndecan, versican, decorin, glypican and/orbiglycan, leads to an improvement in, and to protection of, the humanskin, scalp or mucous membrane. Thus, firming of the skin, increasedelasticity and a better water-binding capacity have been observed, evenafter exposure to UV radiation. The capacity of the skin forregeneration is distinctly improved, so that renewal of the skin andparticularly the healing of wounds take place far more quickly whichalso affords advantages in the treatment of inflammatory skin diseases,more particularly alopecia, cellulitis and roseacea.

Substances which modulate proteoglycans, more particularly lumican,syndecan, versican, decorin, glypican and/or biglycan, may be used ontheir own or in combination with other active principles which

-   -   strengthen dermal macromolecules and make them more resistant to        non-enzymatic glycosylation and thus protect the skin against        toxic environmental poisons and oxidative stress,    -   maintain the balance of the growth factors in aged human skin in        order to improve the renewal and repair of the skin after damage        by UV radiation or in wound healing processes,    -   support the formation of microfibrils in the human skin and        hence provide protection against manifestations of skin ageing,    -   improve the functions of the dermal/epidermal junctions (DEJ)        through improved anchorage by strengthening of the microfibrils,    -   increase the water-binding capacity of the skin and thus        contribute to firmer skin,    -   reduce the formation of microwrinkles and reduce further        wrinkling,    -   delay the appearance of alopecia,    -   reduce skin changes attributable to cellulitis and roseacea,    -   reduce the development of inflammatory processes which lead to        irritation, reddening and itching    -   influence the synthesis of melanin in the skin,    -   strengthen the immune system of the skin and thus improve the        defence system against harmful environmental influences.

Modulators which have proved to be suitable are plant extracts, moreparticularly the extract of Pisum sativum and/or Vigna aconitifolia,extracts of microorganisms and/or fermentation products of vegetableorigin. However, lumican, syndecan, versican, decorin, glypican and/orbiglycan can also be modulated by the application of at least onesubstance selected from the group consisting of mannitol, cyclodextrin,yeast extract and disodium succinate. The combination of theseconstituents in particular leads to an advantageous effect.

Modulation of the molecules by at least one substance selected from thegroup consisting of

-   -   phytosterols such as, for example β-sitosterol, campesterol,        brassicasterol, Δ5-avennasterol, α-spinasterol or stigmasterol;    -   phytoestrogens, such as isoflavones (genistein, daidzein),        stilbenes, lignan;    -   triterpenes, such as lupeol, ursolic acid, arjunolic acid,        oleanolic acid;    -   triterpene saponins and steroid saponins, such as sapogenin,        diosgenin, hecogenin, smilagenin, sarsapogenin, tigogenin,        yamogenin, yuccagenin and bassic acid;    -   peptides, more particularly those which correspond to the growth        factors TGFβ, IL4 and    -   flavonoids and flavonoid derivatives.

Besides these substances or plant extracts, the cosmetic preparationsmay also contain UV protection factors and/or antioxidants. Thecombination of substances which modulate lumican and/or syndecan and/orversican and/or decorin and/or glypican and/or biglycan with UVprotection factors and/or antioxidants leads through the variousmechanisms to a synergistic mode of action and affords excellentprotection against harmful effects and ageing of the skin by UV light.

Lumican

Lumican belongs to the family of keratan sulfates, a group ofleucine-rich proteoglycans (LRP), which are located in the skin togetherwith fibrillar collagen. In the fibrils of various connective tissues,lumican contributes to the elasticity and stability of the tissue.Histologically, collagen strands appear disorganized and withoutstrength in lumican-poor tissue. In the subcutaneous tissue inparticular, there is a lack of order and orientation of collagen strandsand fibroblasts. The histological anomalies can also be verified with anelectron microscope by examining the organization of the collagenmatrix, enlarged interfibrillar spaces and modified morphology of thefibrils.

Syndecan

Syndecans belong to the group of transmembranal heparan sulfateproteoglycans which act as co-receptors with integrin and growth factortyrosinekinase receptors. Through their negative surface charge, theybind to positively charged matrix sections and form firmly anchoredcontacts in the basal lamina. Syndecan-4 is an important surfacereceptor for wound healing and angiogenesis. Syndecan-1 is the mainheparan sulfate proteoglycan of the epidermis. It contains heparansulfate and chondroitin sulfate and is found in mature, fully developedtissue, mainly in simple and layer-form epithelia. In the epidermis,syndecan-1 is found in particular in the suprabasal cell layer, Laminarara. It has a significant share in the migration and proliferation ofkeratinocytes in wound healing. Scientists have recently found anenhancer in syndecan-1 (FiRE—fibroblast growth factor-inducible responseelement—in the promoter of the syndecan-1 gene) which activates geneexpression in keratinocytes at the edges of wounds. They were able toshow that the composition of the extracellular matrix and theavailability of growth factors are influenced by the epidermalregulation of syndecan-1 expression, so that FiRE is a new goal for thegene regulation of the extracellular matrix.

Versican

Versican is one of the large chondroitin sulfate proteoglycans which arefound in the Lamina densa, the middle part of the basal membrane, and inthe Lamina fibroreticularis.

Decorin

Decorin is one of the smallest proteoglycans of the leucine-rich family(LRP). A dermatan sulfate proteoglycan which is largely found in theextracellular matrix of collagen-rich tissue where it covers the surfaceof collagen fibrils.

Glypican

Glypican is a proteoglycan which is anchored by linkage to membranethrough glycosyl phosphatidyl inositol and which binds antithrombin andlipoprotein lipases on the endothelial cell surface.

Biglycan

Biglycan is a small proteoglycan which is released through mesenchymalcells. It is also synthesized by endothelial and epithelial cells and isfound mainly in the pericellular gap, but also in the cell core. Somefactors, such as TGFβ and b-FGF, regulate biglycan synthesis byfibroblasts. The function of biglycan is still largely unknown.

UV Protection Factors and Antioxidants

UV protection factors in the context of the invention are, for example,organic substances (light filters) which are liquid or crystalline atroom temperature and which are capable of absorbing ultravioletradiation and of releasing the energy absorbed in the form oflonger-wave radiation, for example heat. UV-B filters can be oil-solubleor water-soluble. The following are examples of oil-soluble substances:

-   -   3-benzylidene camphor or 3-benzylidene norcamphor and        derivatives thereof, for example        3-(4-methylbenzylidene)-camphor;    -   4-aminobenzoic acid derivatives, preferably        4-(dimethylamino)-benzoic acid-2-ethylhexyl ester,        4-(dimethylamino)-benzoic acid-2-octyl ester and        4-(dimethylamino)-benzoic acid amyl ester;    -   esters of cinnamic acid, preferably 4-methoxycinnamic        acid-2-ethylhexyl ester, 4-methoxycinnamic acid propyl ester,        4-methoxycinnamic acid isoamyl ester, 2-cyano-3,3-phenylcinnamic        acid-2-ethylhexyl ester (Octocrylene);    -   esters of salicylic acid, preferably salicylic acid-2-ethylhexyl        ester, salicylic acid-4-isopropylbenzyl ester, salicylic acid        homomenthyl ester;    -   derivatives of benzophenone, preferably        2-hydroxy-4-methoxybenzo-phenone,        2-hydroxy-4-methoxy-4′-methylbenzophenone,        2,2′-dihydroxy-4-methoxybenzophenone;    -   esters of benzalmalonic acid, preferably 4-methoxybenzalmalonic        acid di-2-ethylhexyl ester;    -   triazine derivatives such as, for example,        2,4,6-trianilino-(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine        and Octyl Triazone or Dioctyl Butamido Triazone (Uvasorb® HEB);    -   propane-1,3-diones such as, for example,        1-(4-tert.butylphenyl)-3-(4′-methoxyphenyl)-propane-1,3-dione;    -   ketotricyclo(5.2.1.0)decane derivatives.

Suitable water-soluble substances are

-   -   2-phenylbenzimidazole-5-sulfonic acid and alkali metal, alkaline        earth metal, ammonium, alkylammonium, alkanolammonium and        glucammonium salts thereof;    -   sulfonic acid derivatives of benzophenones, preferably        2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and salts        thereof;    -   sulfonic acid derivatives of 3-benzylidene camphor such as, for        example, 4-(2-oxo-3-bornylidenemethyl)-benzene sulfonic acid and        2-methyl-5-(2-oxo-3-bornylidene)-sulfonic acid and salts        thereof.

Typical UV-A filters are, in particular, derivatives of benzoyl methanesuch as, for example,1-(4′-tert.butylphenyl)-3-(4′-methoxyphenyl)-propane-1,3-dione,4-tert.butyl-4′-methoxydibenzoyl methane (Parsol 1789) or1-phenyl-3-(4′-isopropylphenyl)-propane-1,3-dione and enamine compounds.The UV-A and UV-B filters may of course also be used in the form ofmixtures. Particularly favorable combinations consist of the derivativesof benzoyl methane, for example 4-tert.butyl-4′-methoxydibenzoylmethane(Parsol® 1789) and 2-cyano-3,3-phenylcinnamic acid-2-ethyl hexyl ester(Octocrylene) in combination with esters of cinnamic acid, preferably4-methoxycinnamic acid-2-ethyl hexyl ester and/or 4-methoxycinnamic acidpropyl ester and/or 4-methoxycinnamic acid isoamyl ester. Combinationssuch as these are advantageously combined with water-soluble filterssuch as, for example, 2-phenylbenzimidazole-5-sulfonic acid and alkalimetal, alkaline earth metal, ammonium, alkylammonium, alkanolammoniumand glucammonium salts thereof.

Besides the soluble substances mentioned, insoluble light-blockingpigments, i.e. finely dispersed metal oxides or salts, may also be usedfor this purpose. Examples of suitable metal oxides are, in particular,zinc oxide and titanium dioxide and also oxides of iron, zirconium,silicon, manganese, aluminium and cerium and mixtures thereof. Silicates(talcum), barium sulfate and zinc stearate may be used as salts. Theoxides and salts are used in the form of the pigments for skin-care andskin-protecting emulsions and decorative cosmetics. The particles shouldhave a mean diameter of less than 100 nm, preferably between 5 and 50 nmand more preferably between 15 and 30 nm. They may be spherical in shapealthough ellipsoidal particles or other non-spherical particles may alsobe used. The pigments may also be surface-treated, i.e. hydrophilicizedor hydrophobicized. Typical examples are coated titanium dioxides, forexample Titandioxid T 805 (Degussa) and Eusolex® T2000 (Merck). Suitablehydrophobic coating materials are, above all, silicones and, amongthese, especially trialkoxyoctylsilanes or simethicones. So-calledmicro- or nanopigments are preferably used in sun protection products.Micronized zinc oxide is preferably used.

Besides the two groups of primary sun protection factors mentionedabove, secondary sun protection factors of the antioxidant type may alsobe used. Secondary sun protection factors of the antioxidant typeinterrupt the photochemical reaction chain which is initiated when UVrays penetrate into the skin. Typical examples are amino acids (forexample glycine, histidine, tyrosine, tryptophane) and derivativesthereof, imidazoles (for example urocanic acid) and derivatives thereof,peptides, such as D,L-carnosine, D-carnosine, L-carnosine andderivatives thereof (for example anserine), carotinoids, carotenes (forexample α-carotene, β-carotene, lycopene) and derivatives thereof,chlorogenic acid and derivatives thereof, liponic acid and derivativesthereof (for example dihydroliponic acid), aurothioglucose,propylthiouracil and other thiols (for example thioredoxine,glutathione, cysteine, cystine, cystamine and glycosyl, N-acetyl,methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl,γ-linoleyl, cholesteryl and glyceryl esters thereof) and their salts,dilaurylthiodipropionate, distearylthiodipropionate, thiodipropionicacid and derivatives thereof (esters, ethers, peptides, lipids,nucleotides, nucleosides and salts) and sulfoximine compounds (forexample butionine sulfoximines, homocysteine sulfoximine, butioninesulfones, penta-, hexa- and hepta-thionine sulfoximine) in very smallcompatible dosages (for example pmole to μmole/kg), also (metal)chelators (for example α-hydroxyfatty acids, palmitic acid, phytic acid,lactoferrine), α-hydroxy acids (for example citric acid, lactic acid,malic acid), humic acid, bile acid, bile extracts, bilirubin,biliverdin, EDTA, EGTA and derivatives thereof, unsaturated fatty acidsand derivatives thereof (for example γ-linolenic acid, linoleic acid,oleic acid), folic acid and derivatives thereof, ubiquinone andubiquinol and derivatives thereof, vitamin C and derivatives thereof(for example ascorbyl palmitate, Mg ascorbyl phosphate, ascorbylacetate), tocopherols and derivatives (for example vitamin E acetate),vitamin A and derivatives (vitamin A palmitate) and coniferyl benzoateof benzoin resin, rutinic acid and derivatives thereof, α-glycosylrutin, ferulic acid, furfurylidene glucitol, carnosine, butylhydroxytoluene, butyl hydroxyanisole, nordihydroguaiac resin acid,nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid andderivatives thereof, mannose and derivatives thereof,Superoxid-Dismutase, zinc and derivatives thereof (for example ZnO,ZnSO₄), selenium and derivatives thereof (for example seleniummethionine), stilbenes and derivatives thereof (for example stilbeneoxide, trans-stilbene oxide) and derivatives of these active substancessuitable for the purposes of the invention (salts, esters, ethers,sugars, nucleotides, nucleosides, peptides and lipids).

EXAMPLES

Glucosaminoglycans and proteoglycans of human skin varying in age wereinvestigated to show that ageing-induced changes of proteoglycanscontribute to the corresponding appearance of the skin and to itsmechanical properties. To this end, samples of child's skin, adult'sskin and aged skin were taken and cell cultures of keratinocytes andfibroblasts were cultivated from them.

In order to determine the features of human skin as a function of age,the following classical techniques were used [19-WEGROWSKI, Y.; PALTOT,V.; GILLERY, P.; KALIS, B.; RANDOUX, A.; MAQUART, F. X.; BiochemicalJournal 2995, 307, 3, 673-678; WEGROWSKI, Y.; GILLERY, P.; KOTLARZ, G.;PERREAU, C.; GEORGES, N. and MAQUART, F. X.; Molecular and CellularBiochemistry, 200, 205, 125-131].

For glucosaminoglycans:

Use of radioactively marked molecules, such as tritium-markedglucosamines, for all glucosaminoglycans except keratan sulfate and ³⁵Sfor all sulfated glucosaminoglycans. Quantitative determination of themarked molecules in glucosaminoglycans and electrophoresis techniques.

For proteoglycans:

Northern Blot technique for determining the RNA messengers in cells oflow molecular weight proteoglycans, such as lumican and syndecan.

1. Investigation of Human Dermal Fibroblasts from Donors in DifferentAge Categories

The quantity of RNA messengers of the proteoglycan lumican wasdetermined in a fibroblast culture by the Northern Blot technique. Theresults are set out in Table 1 as the ratio of the quantity of RNAmessengers for lumican to the quantity of ¹⁸S ribosomal RNA. TABLE 1Ratio of the quantity of RNA messengers for lumican to the quantity of¹⁸S ribosomal RNA for different age categories Age No. of Mean value of(years) volunteers [lumican RNAm]:[18S ribosomal RNA] ratio  1-15 8 4.016-50 8 2.7 51-71 8 1.9

The data in Table 1 clearly show that the quantity of RNA messengers forlumican decreases with increasing age of the fibroblast donor. This canmean that the lumican synthesis rate in the skin cells has a significantinfluence on the appearance of ageing skin.

2. Investigation of Human Keratinocytes from Donors in Different AgeCategories

The quantity of RNA messengers of the proteoglycan syndecan-1 wasdetermined in a keratinocyte culture by the Northern Blot technique. Theresults are set out in Table 2 as the ratio of the quantity of RNAmessengers for syndecan-1 to the quantity of ¹⁸S ribosomal RNA. TABLE 2Ratio of the quantity of RNA messengers for syndecan-1 to the quantityof ¹⁸S ribosomal RNA for different age categories Age No. of Mean valueof (years) volunteers [syndecan-1 RNAm]:[18S ribosomal RNA] ratio  1-154 5.1 16-50 4 3.2 51-71 4 3.1

Even comparable investigation of the proteoglycan syndecan-1 shows areduction in the RNA messengers with increasing age of the keratinocytedonors, so that it must be assumed that there is a reduction in thesynthesis of this proteoglycan. This is also an indication that thesynthesis rate of the proteoglycans can have a significant influence onthe ageing of skin.

3. Stimulation of the Lumican mRNA Content in MRC5 Fibroblasts by IGF-1.

The stimulation of the lumican mRNA content in MRC5 fibroblastsincubated with increasing concentrations of the growth factor IGF-1(Insulin-like Growth Factor 1, Sigma-Aldrich) was investigated. Thecontent of mRNA of the proteoglycan lumican in cultures of humanfibroblasts—MRC5 fibroblasts—was determined by the Northern Blot method.TABLE 3 Results as lumican mRNA content against housekeeping gene 36B4mRNA as a function of the IGF-1 concentration Mean value Concentrationof IGF-1 (ng/ml) [mRNA lumican/36B4 mRNA] 0 0.86 0.1 1.38 1 2 10 3.2

The results clearly show that IGF-1 in cultures of MRC5 fibroblastsincreases the mRNA content for lumican.

1-17. (canceled)
 18. A method of protecting the human skin againstaging, said method comprising applying to the skin a compositioncontaining at least one substance which modulates at least oneproteoglycan selected from a group consisting of lumican, syndecan,versican, decorin, glypican and biglycan.
 19. A method according toclaim 18 wherein the at least one substance modulates lumican, syndecanor decorin.
 20. A method according to claim 19 wherein the at least onesubstance is selected from at least one plant extract.
 21. A methodaccording to claim 20 wherein the at least one plant extract is selectedfrom an extract of Pisum sativum and Vigna aconitifolia.
 22. A methodaccording to claim 18 wherein the at least one substance is selectedfrom a group consisting of mannitol, cyclodextrin, yeast extract anddisodium succinate.
 23. A method according to claim 18 wherein the atleast one substance is selected from a group consisting of phytosterols,triterpenes, triterpene saponins, TGFβ, IL4, flavonoids, and flavonoidderivatives.
 24. A method of improving the regeneration and healing ofthe human skin, said method comprising applying to the skin acomposition containing at least one substance which modulates at leastone proteoglycan selected from a group consisting of lumican, syndecan,versican, decorin, glypican and biglycan.
 25. A method according toclaim 24 wherein the at least one substance modulates lumican, syndecanor biglycan.
 26. A method according to claim 24 wherein the at least onesubstance is selected from at least one plant extract.
 27. A methodaccording to claim 26 wherein the at least one plant extract is selectedfrom an extract of Pisum sativum and Vigna aconitifolia.
 28. A methodaccording to claim 24 wherein the at least one substance is selectedfrom a group consisting of mannitol, cyclodextrin, yeast extract anddisodium succinate.
 29. A method according to claim 24 wherein the atleast one substance is selected from a group consisting of phytosterols,triterpenes, triterpene saponins, TGFβ, IL4, b-FGF, flavonoids, andflavonoid derivatives.
 30. A method of increasing the mRNA content forlumican, said method comprising incubating MRC5 fibroblasts with IGF-1.